Electrophoretic separation is caused by application of a voltage. Charged particles migrate in the electric field to the corresponding poles, for example negatively charged particles to the positive pole. In this process, each charged particle exhibits different speeds in the electric field because of different mobilities. The mobility depends on the charge number and on the radius of the particle and on the hydrate layer forming on the particle. The viscosity of the buffer likewise impairs the mobility of the charged particles. Charged particles with different mobility constants can therefore be separated from one another.
A further electrophoretic effect which influences the separation in the electric field is the electroosmotic flow (EOF). Said uniform and directional flow is generated by surface charges on the inner capillary surface. Capillary materials with a high charge density generate a high EOF. A negative surface such as is formed in the case of glass, for example, produces a flow in the direction of the positive pole. The EOF accelerates negatively charged particles and brakes positively charged particles. Neutral particles, by contrast, migrate through the capillary with the EOF. The pH has a significant influence on the surface charge, and therefore likewise on the EOF.
Glass capillaries are routinely used, since they can be produced simply and cost-effectively. A further great advantage is the optical transparency to light in the UV/VIS region. Optical detectors can therefore be used on-column without coming into contact with the liquid subjected to voltage. However, it is only compounds which absorb in the appropriate wavelength region which can be detected. Thus, for example, monosaccharides and oligosaccharides have no chromophore. Other sensitive detectors such as mass-selective and electrochemical detectors must be used for these compounds. However, said detectors come into contact with the liquid subjected to voltage, which leads to a significant deterioration or to the failure of these detectors. There is thus a mandatory requirement to remove the voltage from the separation section before the fluid reaches the detector.
A further disadvantage of glass capillaries is the high adsorption tendency of compounds which can agglomerate irreversibly on the negative surface. An inertization of the inner surface can be brought about by a thin coating with a polymer. However, the intensive UV radiation of the detector can easily irreversibly damage the fine polymer layer.
To date, the problem of downstream detection has been solved by a so-called “sheath interface”, for example. After the electrophoretic separation, the fluid passes to the mass spectrometer via an interface. A liquid (sheath liquid) is fed in the interface in order to be able to remove the voltage from the capillary. An undesired effect of the feed is the dilution with the fluid from the capillary. This substantially reduces the detection sensitivity.
In order not to worsen the detection sensitivity in the downstream detection, attempts have been made in recent developments to dispense with the sheath liquid and, instead, to use a so-called “sheathless interface”. Novel developments are described by Zamfir et al. in Journal of Chromatography A, 1159 (2007), 2-13. Conductive so-called emitters consist of a specifically produced glass capillary and a conductive material which is applied to the outside of the glass capillary. The emitter is then connected to the separation capillary and electrical contact is established. The requirements placed on the design and material of the emitter are very stringent for the purpose of obtaining an excellent and reproducible spray characteristic for mass spectrometry. Furthermore, the emitter must not have a negative influence on the separation quality and thus on the analytical performance. The design has the advantage that the emitter can be applied flexibly to various separation columns, although separation capillary and emitter must be cleanly connected to one another. According to the publication, however, there are difficulties in implementing said requirements.
In a workshop for capillary electrophoresis which was held by Beckman/Coulter and took place in September 2009 in Basel, a newly-developed emitter was presented. In this case, the last 4 cm of a glass capillary are etched at one end until the wall of the glass capillary becomes porous and the voltage can thereby be removed from the capillary. The porous part is inserted into a metal housing (=electrode) and electrical contact is established. A conductive liquid is flushed between porous capillary and metal housing in order to transport away gas bubbles that have formed, this being done by electrolysis of the aqueous buffer at the electrode. However, the porous part of the capillary can lead to a much higher surface adsorption of compounds, and this can result in substantial worsening of the analytical performance. The very shock-sensitive design of the treated capillary can also be disadvantageous.
A further known approach consists in the application of chip technology. In “Miniaturization of Analytical Systems”, ISBN-10: 0-470-06110-3, page 237, A. Rios et al. describe solutions on microchips with integrated capillary electrophoresis which enable the voltage to be removed from the separation section before the detection.
In chip technology, the term separation capillary is replaced by micro separation channel, micro separation channels being introduced into the chip by etching processes.
The above publication presents a variant which has a side arm on a chip which departs from the actual separation channel, in which side arm the second contact electrode is also situated. Said side arm is coated with polyacrylamide in order to substantially reduce the electroosmotic flow. The separation channel made from glass, by contrast, is not coated, and so a higher EOF is achieved. Said difference in the EOF leads to an indirect hydrodynamic flow downstream of the bifurcation as far as the channel end. This flow is supported by an increase in the flow resistance in the side arm by virtue of the fact that the length of the side arm is larger than the distance from the bifurcation as far as the channel end. Mass spectrometry is described here as the detection technique. In the case of electrochemical detections, use is made of the term “off-channel detection” when the voltage is to be removed before the electrochemical detection. Several variants are presented with the aid of a decoupler in the above publication and by H. Chen et al. in Trends in Analytical Chemistry, Volume 26, No. 2, 2007.
J. S. Rossier et al., Journal of Electroanalytical Chemistry, 492 (2000), 15 describes a design in which microholes consisting of another polymer material are integrated at the end of the separation channel. This design allows the voltage to be removed from the separation channel before the electrochemical detection. Osbourne et al., Analytical Chemistry, 75 (2003), 2710, likewise describe a design with holes at the end of the separation channel. The holes are closed with a cellulose acetate membrane. The membrane is porous enough for electrical contact to be made with the electrode.
One object of the present invention is to provide a suitable capillary for capillary electrophoresis which enables the use of sensitive, nonoptical detectors such as, for example, mass-selective or electrochemical detectors, and minimizes the loss of analyte as far as possible (for example by irreversible immobilization).
Further objects of the present invention are to provide a suitable chemical separation and analysis device which includes the inventive capillary, and a chemical separation and analysis method with application of the inventive device.
In accordance with a first aspect of the present invention, this object is achieved by providing a plastic capillary tube for capillary electrophoresis, in which the plastic capillary tube has an inlet opening, an outlet opening and at least one hole in the capillary tube wall and the diameter of the hole on the inside of the capillary tube wall dL(innen) lies in the range from 0.5 μm to 30 μm.
As set forth above, the inventive capillary tube is a polymer or plastic capillary tube, that is to say a capillary tube made from a polymer material.
By contrast with the conventional glass capillary tubes, plastic capillary tubes exhibit fewer instances of surface adsorption for compounds with a strong tendency thereto because of their chemical structure. Examples are proteins and oligosaccharides. Electrophoretic separation in plastic capillary tubes is favourable for such classes of compound. Furthermore, chemically and mechanically stable plastic capillary tubes can be produced more cost-effectively.
As already discussed above, the use of sensitive and selective detectors presupposes that the voltage applied for the electrophoretic separation is removed as effectively as possible before the detector is reached. Within the scope of the present invention, this is enabled by virtue of the fact that the plastic capillary tube has at least one hole in the capillary tube wall and the diameter of the hole on the inside of the capillary tube wall dL(innen) lies in the range from 0.5 μm to 30 μm. The selected diameter of the hole in the capillary wall renders it possible, on the one hand, to remove the voltage but, on the other hand, the fluid is prevented as far as possible from escaping from the hole in the capillary tube wall for given test conditions.